The protein Sex-lethal (SXL) controls dosage compensation in Drosophila by inhibiting splicing and subsequently translation of male-specific-lethal-2 (msl-2) transcripts. We have previously shown that SXL blocks the binding of U2 auxiliary factor (U2AF) to the polypyrimidine (Py)-tract associated with the 3′ splice site of the regulated intron. We now report that a second pyrimidine-rich sequence containing 11 consecutive uridines immediately downstream from the 5′ splice site is required for efficient splicing inhibition by SXL. Psoralen-mediated crosslinking experiments suggest that SXL binding to this uridine-rich sequence inhibits recognition of the 5′ splice site by U1 snRNP in HeLa nuclear extracts. We also show that SXL interferes with the binding of the protein TIA-1 to the uridine-rich stretch. Because TIA-1 binding to this sequence is necessary for U1 snRNP recruitment to msl-2 5′ splice site and for splicing of this pre-mRNA, we propose that SXL antagonizes TIA-1 activity and thus prevents 5′ splice site recognition by U1 snRNP. Taken together with previous data, we conclude that efficient retention of msl-2 intron involves inhibition of early recognition of both splice sites by SXL.

The process of eukaryotic gene expression involves a diverse number of steps including transcription, RNA processing, transport, translation, and mRNA turnover. A critical step in understanding this process will be the development of mathematical models that quantitatively describe and predict the behavior of this complex system. We have simulated eukaryotic mRNA turnover in a linear multicomponent model based on the known mRNA decay pathways in yeast. Using rate constants based on experimental data for the yeast unstable MFA2 and stable PGK1 transcripts, the computational modeling reproduces experimental observations after minor adjustments. Subsequent analysis and a series of in silico experiments led to several conclusions. First, we demonstrate that mRNA half-life as commonly measured underestimates the average life span of an mRNA. Second, due to the properties of the pathways, the measurement of a half-life can predominantly measure different steps in the decay network. A corollary of this fact is that different mRNAs will be affected differentially by changes in specific rate constants. Third, the way to obtain the largest change of levels of mRNA for the smallest changes in rate is by changing the rate of deadenylation, where a large amount of regulation of mRNA decay occurs. Fourth, the 3′-to-5′ degradation of mRNA shows mRNA-specific rates of degradation that are dependent on the 5′ structure of the mRNA. These programs can be run over the Web, are adaptable to other eukaryotes, and provide outputs as graphs and virtual northern gels, which can be directly compared to experimental data. Therefore, this model constitutes a useful tool for the quantitative analysis of the process and control of mRNA degradation in eukaryotic cells.

Translation initiation promoted by picornavirus internal ribosome entry site (IRES) elements is dependent on the association of specific IRES sequences to the initiation factor eIF4G. However the RNA determinants interacting with other components of the translational machinery are still unknown. In this study, we have identified novel RNA–protein interactions between the foot-and-mouth disease virus (FMDV) IRES and three translation initiation factors. A doublet of 116/110 kDa that crosslinked to the FMDV IRES is a component of eIF3. We show here that domain 5 holds the preferential binding site for eIF3, although this complex initiation factor can establish multiple contacts with the IRES structure. We have also identified the phylogenetically conserved hairpin of domain 5 as the RNA motif responsible for eIF4B interaction. Mutation of this stem-loop structure abrogated eIF4B, but not eIF3, binding to the IRES. Remarkably, IRES mutants severely affected in their interaction with eIF4B showed a mild reduction in IRES activity when tested in the context of a bicistronic expression vector in transfected cells. Finally, we provide evidence of the interaction of eIF4GII with FMDV IRES, the RNA determinants for this interaction being shared with its functional homolog eIF4GI. The FMDV Lb protease generated a C-terminal fragment of eIF4GII that binds to the IRES as efficiently as the intact protein. Competition experiments showed that titration of eIF4B or p110/116 interaction with the FMDV IRES required a large excess of competitor relative to eIF4G, strongly suggesting that eIF4G-IRES interaction is a limiting factor to titrate the IRES. Comparative analysis of the activity of IRES mutants affected in domains 4 and 5 regarding their pattern of RNA–protein complex formation demonstrates that while binding of eIF4B with the FMDV IRES is dispensable, interaction of eIF4G is a central feature of the activity of this element.

Protein-dependent group II intron splicing provides a forum for exploring the roles of proteins in facilitating RNA-catalyzed reactions. The maize nuclear gene crs1 is required for the splicing of the group II intron in the chloroplast atpF gene. Here we report the molecular cloning of the crs1 gene and an initial biochemical characterization of its gene product. Several observations support the notion that CRS1 is a bona fide group II intron splicing factor. First, CRS1 is found in a ribonucleoprotein complex in the chloroplast, and cofractionation data provide evidence that this complex includes atpF intron RNA. Second, CRS1 is highly basic and includes a repeated domain with features suggestive of a novel RNA-binding domain. This domain is related to a conserved free-standing open reading frame of unknown function found in both the eubacteria and archaea. crs1 is the founding member of a gene family in plants that was derived by duplication and divergence of this primitive gene. In addition to its previously established role in atpF intron splicing, new genetic data implicate crs1 in chloroplast translation. The chloroplast splicing and translation functions of crs1 may be mediated by the distinct protein products of two crs1 mRNA forms that result from alternative splicing of the crs1 pre-mRNA.

P element somatic inhibitor (PSI) is a 97-kDa RNA-binding protein with four KH motifs that is involved in the inhibition of splicing of the Drosophila P element third intron (IVS3) in somatic cells. PSI interacts with a negative regulatory element in the IVS3 5′ exon. This element contains two pseudo-5′ splice sites, termed F1 and F2. To identify high affinity binding sites for the PSI protein, in vitro selection (SELEX) was performed using a random RNA oligonucleotide pool. Alignment of high affinity PSI-binding RNAs revealed a degenerate consensus sequence consisting of a short core motif of CUU flanked by alternative purines and pyrimidines. Interestingly, this sequence resembles the F2 pseudo-5′ splice site in the P element negative regulatory element. Additionally, a negative in vitro selection of PCR-mutagenized P element 5′ exon regulatory element RNAs identified two U residues in the F1 and F2 pseudo-5′ splice sites as important nucleotides for PSI binding and the U residue in the F2 region is a nearly invariant nucleotide in the consensus SELEX motif. The high affinity PSI SELEX sequence acted as a splicing inhibitor when placed in the context of a P element splicing pre-mRNA in vitro. Data from in vitro splicing assays, UV crosslinking and RNA-binding competition experiments indicates a strong correlation between the binding affinities of PSI for the SELEX sequences and their ability to modulate splicing of P element IVS3 in vitro.

Maturation of 18S rRNA and biogenesis of the 40S ribosomes in yeast requires a large number of trans-acting factors, including the U3 small nucleolar ribonucleoprotein (U3 snoRNP), and the recently characterized cyclase-like protein Rcl1p. U3 snoRNP is a key particle orchestrating early 35S rRNA cleavage events. A unique property of Rcl1p is that it specifically associates with U3 snoRNP, but this association appears to occur only at the level of nascent ribosomes and not with the U3 monoparticle. Here we report the characterization of Bms1p, a protein that associates with Rcl1p in multiple structures, including a specific complex sedimenting at around 10S. Like Rcl1p, Bms1p is an essential, evolutionarily conserved, nucleolar protein, and its depletion interferes with processing of the 35S pre-rRNA at sites A0, A1, and A2, and the formation of 40S subunits. The N-terminal domain of Bms1p has structural features found in regulatory GTPases and we demonstrate that mutations of amino acids implicated in GTP/GDP binding affect Bms1p activity in vivo. The results indicate that Bms1p may act as a molecular switch during maturation of the 40S ribosomal subunit in the nucleolus.

Bms1p and Tsr1p define a novel family of proteins required for synthesis of 40S ribosomal subunits in Saccharomyces cerevisiae. Both are essential and localize to the nucleolus. Tsr1p shares two extended regions of similarity with Bms1p, but the two proteins function at different steps in 40S ribosome maturation. Inactivation of Bms1p blocks at an early step, leading to disappearance of 20S and 18S rRNA precursors. Also, slight accumulation of an aberrant 23S product and significant 35S accumulation are observed, indicating that pre-rRNA processing at sites A0, A1, and A2 is inhibited. In contrast, depletion of Tsr1p results in accumulation of 20S rRNA. Because processing of 20S to 18S rRNA occurs in the cytoplasm, this suggests that Tsr1p is required for assembly of a transport- or maturation-competent particle or is specifically required for transport of 43S pre-ribosomal particles, but not 60S ribosome precursors, from the nucleus to the cytosol. Finally, Bms1p is a GTP-binding protein, the first found to function in ribosome assembly or rRNA processing.

The SRPK family of kinases is specific for RS domain-containing splicing factors and known to play a critical role in protein–protein interaction and intracellular distribution of their substrates in both yeast and mammalian cells. However, the function of these kinases in pre-mRNA splicing remains unclear. Here we report that SKY1, a SRPK family member in Saccharomyces cerevisiae, genetically interacts with PRP8 and PRP17/SLU4, both of which are involved in splice site selection during pre-mRNA splicing. Prp8 is essential for splicing and is known to interact with both 5′ and 3′ splice sites in the spliceosomal catalytic center, whereas Prp17/Slu4 is nonessential and is required only for efficient recognition of the 3′ splice site. Interestingly, deletion of SKY1 was synthetically lethal with all prp17 mutants tested, but only with specific prp8 alleles in a domain implicated in governing fidelity of 3′AG recognition. Indeed, deletion of SKY1 specifically suppressed 3′AG mutations in ACT1-CUP1 splicing reporters. These results suggest for the first time that 3′AG recognition may be subject to phosphorylation regulation by Sky1p during pre-mRNA splicing.

Association of U2 snRNP with the pre-mRNA branch region is the first ATP-dependent step in spliceosome assembly. The basis of this energy dependence is not known. Previously, we identified minimal intron-derived substrates that form complexes with U2 independent of ATP. Here, we identify the intron region linked to the ATP dependence of this step by comparing these substrates to longer RNAs that recapitulate the ATP requirement. This region needed to impose ATP dependence lies immediately 5′ to the branch site. Sequences ranging from 6 to 14 nt yield a near linear inhibitory effect on efficiency of complex formation with U2 snRNP, with 18 nt yielding near maximal ATP dependence. This region is not protected prior to U2 addition, and RNase H targeting of the region within nuclear extract converts an ATP-dependent substrate into an ATP-independent one. Within this region, there is no sequence specificity linked with the ATP requirement, as neither a specific sequence is needed, nor even nucleobases. These data and the results of other modifications suggest models in which the 18-nt region is a target for interactions with U2 snRNP in an ATP-bound or -activated conformation.

DNA harvested directly from complex natural microbial communities by PCR has been successfully used to predict RNase P RNA structure, and can potentially provide an abundant source of information for structural predictions of other RNAs. In this study, we utilized genetic variation in natural communities to test and refine the secondary and tertiary structural model for the bacterial tmRNA. The variability of proposed tmRNA secondary structures in different organisms and the lack of any predicted tertiary structure suggested that further refinement of the tmRNA could be useful. To increase the phylogenetic representation of tmRNA sequences, and thereby provide additional data for statistical comparative analysis, we amplified, sequenced, and compared tmRNA sequences from natural microbial communities. Using primers designed from gamma proteobacterial sequences, we determined 44 new tmRNA sequences from a variety of environmental DNA samples. Covariation analyses of these sequences, along with sequences from cultured organisms, confirmed most of the proposed tmRNA model but also provided evidence for a new tertiary interaction. This approach of gathering sequence information from natural microbial communities seems generally applicable in RNA structural analysis.

Ribosome synthesis is a highly complex process and constitutes a major cellular activity. The biogenesis of this ribonucleoprotein assembly requires a multitude of protein trans-acting factors including several putative ATP-dependent RNA helicases of the DEAD-box and related protein families. Here we show that the previously uncharacterized Saccharomyces cerevisiae open reading frame YLR276C, hereafter named DBP9 (DEAD-box protein 9), encodes an essential nucleolar protein involved in 60S-ribosomal-subunit biogenesis. Genetic depletion of Dbp9p results in a deficit in 60S ribosomal subunits and the appearance of half-mer polysomes. This terminal phenotype is likely due to the instability of early pre-ribosomal particles, as evidenced by the low steady-state levels and the decreased synthesis of the 27S precursors to mature 25S and 5.8S rRNAs. In agreement with a role of Dbp9p in 60S subunit synthesis, we find that increased Dbp9p dosage efficiently suppresses certain dbp6 alleles and that dbp6/dbp9 double mutants show synthetic lethality. Furthermore, Dbp6p and Dbp9p weakly interact in a yeast two-hybrid assay. Altogether, our findings indicate an intimate functional interaction between Dbp6p and Dbp9p during the process of 60S-ribosomal-subunit assembly.

Guide RNAs (gRNAs) are encoded both in the maxicircle and minicircle components of the mitochondrial DNA of trypanosomatid protozoa. These RNAs mediate the precise insertion and deletion of U residues in transcripts of the maxicircle DNA. We showed previously that the old UC laboratory strain of Leishmania tarentolae apparently lost more than 40 minicircle-encoded gRNAs that are present in the recently isolated LEM125 strain [Thiemann et al., EMBO J, 1994, 13:5689–5700]. We have further analyzed the population of minicircle-encoded gRNAs in the LEM125 strain. Sau3AI and MspI minicircle libraries were constructed and screened for novel gRNAs by negative colony hybridization. This search yielded 20 minicircles encoding new gRNAs that covered most of the remaining gaps in the editing cascades of the ND8, ND9, G4, and G5 genes, and in addition, more than 30 minicircles containing either unassigned or undetectable gRNA genes. We also completely sequenced 34 of the 45 minicircle sequence classes encoding previously identified gRNAs. A total of 19 pairs of redundant gRNAs, which are gRNAs of different sequences covering the same editing blocks, were identified. The gRNAs in each redundant pair generally had different relative abundances and different extents of mismatches with edited sequences. Alignments of the minicircles encoding redundant gRNAs yielded 59 to 93% matching nucleotides, suggesting an origin from duplication of ancestral minicircles and subsequent genetic drift. We propose a functional explanation for the existence of redundant gRNAs in this strain.

AU-rich elements (AREs) located in the 3′ UTRs of the messenger RNAs (mRNAs) of many mammalian early response genes promote rapid mRNA turnover. HuR, an RRM-containing RNA-binding protein, specifically interacts with AREs, stabilizing these mRNAs. HuR is primarily nucleoplasmic, but shuttles between the nucleus and the cytoplasm via a domain called HNS located between RRM2 and RRM3. We recently showed that HuR interacts with two protein ligands, pp32 and APRIL, which are also shuttling proteins, but rely on NES domains recognized by CRM1 for export. Here we show that heat shock induces increased association of HuR with pp32 and APRIL through protein–protein interactions and that these ligands partially colocalize with HuR in cytoplasmic foci. HuR associations with the hnRNP complex also increase, but through RNA links. CRM1 coimmunoprecipitates with HuR only after heat shock, and nuclear export of HuR becomes sensitive to leptomycin B, an inhibitor of CRM1. Export after heat shock requires the same domains of HuR (HNS and RRM3) that are essential for binding pp32 and APRIL. In situ hybridization and coimmunoprecipitation experiments show that LMB treatment blocks both hsp70 mRNA nuclear export and its cytoplasmic interaction with HuR after heat shock. Together, our results argue that upon heat shock, HuR switches its export pathway to that of its ligands pp32 and APRIL, which involves the nuclear export factor CRM1. HuR and its ligands may be instrumental in the nuclear export of heat-shock mRNAs.